Dye intermediate and method

ABSTRACT

Various classes of dyes are provided having acid, ester or amide groups for covalent linking to biomolecules. The dyes may be prepared by use of a compound of formula (I)                    
     where R 1  comprises a linker and a carboxy including acid, salt, ester including N-hydroxysuccinimide, activated ester or amide group; R 2 , R 3 , R 4  and R 5  are H, C 1 -C 10  alkyl or aralkyl or a group to modify solubility or electronic or spectral properties or a functional linking group: or R 4 -R 5  and/or R 2 -R 4  and/or R 2 -R 3  are linked to form an extended ring system; and R 6  is H or CHO or NO.

Dyes have applications in many technologies, both new and wellestablished, which count amongst them textile dyeing, optical datastorage media and various detection methods, for both chemical andbiological use. Within these applications various properties of the dyesare exploited in their actual detection which may ultimately be tracedback to absorption, reflectance and luminescence (chemiluminescence,fluorescence and phosphorescence). However, a recurring theme of manyapplications is the need to form a relatively permanent link, in theform of a covalent bond, between the dye and the substrate of choice.The substrate may be inter alia: a relatively inert surface or probepossibly of insoluble polymeric origin; a macromolecule of biologicalorigin e.g. a protein, antibody or oligonucleotide; or a small moleculeof either biological or synthetic origin e.g. a hapten for use indirected or molecular recognition based applications; or a moleculewhich may modify the properties of the dye e.g. a water solubilisinggroup such as a carbohydrate or other polar residues; or a species whichprotects the dyes from or decreases the rate of photofading of the dye,such as hindered amines and various transition metal complexes; orpossibly another dye conjugate system thus enabling the possibility ofenergy transfer or fluorescence quenching based applications. Therequirement for a covalent linkage means that many commercial products,spanning a large range of dye classes and possessing the desiredspectral properties, cannot be used. This therefore necessitates eitherthe modification of an existing product or the complete synthesis fromappropriate materials such that a suitable grouping is available forlinking the dye and substrate. There are many different sorts of linkageknown, the main requirements being that the linkage is relatively stableand that it is easily introduced in high yield or efficiency. One of themost versatile groupings is that of the carboxylic acid which is readilyconverted to a range of reactive species or used to form a link toanother substance containing the reactive linking species of choice.

This invention is concerned with a relatively simple and easily prepareddye intermediate and its use for preparing a range of dyes particularlyfluorescent dyes having reactive groups by which they can be linked tovarious substrates. The intermediate is a m-aminophenol having theformula (I)

Where

R¹ is —R^(a)R^(b), where R^(a) is C₁-C₁₀ alkylene or aralkylene whichmay optionally contain 1 to 4 oxygen, nitrogen or sulphur atoms in astraight or branched chain, and R^(b) is carboxy including acid, issalt, ester including N-hydroxysuccinimide, activated ester or amide,

R², R³, R⁴ and R⁵ are the same or different and each is: H; or C₁-C₁₀alkyl or aralkyl which may optionally contain 1 to 4 oxygen, nitrogen orsulphur atoms in a straight or branched chain; or R^(c) where R^(c) is agroup to modify solubility, or a group to modify electronic and/orspectral properties, or a functional linking group; or —R^(a)R^(c)wherein R^(a) and R^(c) are herein defined,

or R⁴-R⁵ and/or R²-R⁴ and/or R²-R³ are linked to form an extended ringsystem, carbocyclic or heterocyclic, aromatic or aliphatic which isunsubstituted or substituted as above,

R⁶ is H or —CR⁹O or N—O.

R⁹ is H, C₁-C₆ alkyl phenyl or R^(a)R^(c) wherein R^(a) and R^(c) areherein defined.

R¹ may be different or the same as R².

R², R³, R⁴ and R⁵ may contain carbon chains which are linked to formextended ring systems, either aliphatic or aromatic, for example

where

each of R⁷ and R⁸ are defined as R², R³, R⁴ and R⁵.

Many of the dyes described below are made from compounds of formula (I)in which R⁶ has been changed from H to either formyl or nitroso, forexample

These may be made from the corresponding compounds where R⁶ is H bystandard methods.

Groups to modify solubility include phosphate, sulphonate, carbohydrate,poly(oxyethylene) and perfluoroalkyl. Groups to modify electronic and/orspectral properties include nitro, cyano, halogen and alkoxy. Functionalgroups for linking to another component include carboxylic acid orderivative or activated ester, azide, amine, hydroxyl, sulphonylchloride, isothiocyanate, phosphoramidite, vinyl sulphone, maleimide,halotriazine, iodoacetamide and N-hydroxysuccinimide; see also AndrewGarman: “Non-Radioactive Labelling: A Practical Introduction” publishedby Academic Press, London, 1997, and references cited therein.

These compounds may be linked, preferably through R^(b) or alternativelythrough R², R³, R⁴, R⁵, R⁷ or R⁸ to a support such as polyethyleneglycol, polyethyleneimine, polysaccharides such as dextran, or aderivatised substrate such as that used in solid phase synthesis e.g.polystyrene, polyacrylate or glass.

Preferably R^(a) is C₁-C₄ alkylene. Certain of these compounds can bemade by an advantageous process which forms another aspect of thisinvention, and which comprises reacting

with GG¹

where

G is CHR=CR— where R is H or C₁-C₄ alkyl

and G¹ is an electron withdrawing group for example CN or carboxy wherethe carboxy group is acid, salt, ester or amide.

Other compounds may be made by the use of G²R^(a)G¹ where G² is aleaving group and R^(a) and G¹ are herein defined.

An intermediate compound of formula (I) may be immobilised on a supportand there reacted to form the desired dye. This method may give accessto a combinatorial library of dyes or an easy way of purifying dyes, ormay permit the labelling of a first nucleotide or amino acid of a solidphase oligonucleotide or oligopeptide synthesiser.

In one aspect, this invention provides use of the said intermediate tomake a dye selected from a defined group, or a leuco-dye or reducedanalogue of said dye. In another aspect, the invention provides theresulting dyes, and their leucodye analogues, as new compounds. The dyesare as follows (in each case, R¹, R², R³, R⁴ and R⁵ are as defined abovefor the intermediate compound (I); and may be the same or different atdifferent parts of the molecule):

where

X is O, NH or =NCOAr

X¹ is H, CN, CHO, CH=N⁺(R^(d)R^(e)), NO, COOH, COOR^(f), CONR^(g)R^(h),C₁-C₁₀ alkyl, aralkyl or aryl or

where

X³ is NH, N-R^(k), O or S, wherein X¹ is either unsubstituted orsubstituted by: a group to modify solubility; or a group to modifyelectronic and/or spectral properties; or a functional group for linkingto another component,

R^(d) and R^(e) are alkyl, aryl, aralkyl,

R^(f), R^(g), R^(h) and R^(k) are R^(a)R^(c) wherein R^(a) and R^(c) areherein defined,

X² is H, CN, OR^(i), Cl, Br, alkyl or aryl which maybe unsubstituted orsubstituted by: a group to modify solubility; or a group to modifyelectronic and/or spectral properties; or a functional group for linkingto another component;

where R^(i) is MeC₆H₄SO₂—, CH₃SO₂—, P=O(OR^(j))₂

R^(j) is alkyl, aryl, aralkyl

X¹ and X² may contain atoms which are linked to form a carbocyclic,heterocyclic, aliphatic or aromatic ring system which may be substitutedor unsubstituted with a group to modify electronic and/or spectralproperties and/or a group to modify solubility properties and/or afunctional linking group;

provided that, when X is O, then X¹ is not H, CN, or C₁-C₁₀ alkyl,aralkyl or aryl; and provided that when X is O, then X³ is not S;

where

Y is CN or CONH₂ or CH₂NH₂,

Y¹ is NH or O or =NCOAr,

Y² and Y⁴ are: H; or C₁-C₁₀ alkyl or aralkyl which may optionallycontain 1 to 4 oxygen, nitrogen or sulphur atom in a straight orbranched chain; or R^(c) where R^(c) is a group to modify solubility, ora group to modify electronic and/or spectral properties, or a functionallinking group; or —R^(a)R^(c) wherein R^(a) and R^(c) are hereindefined,

Y³ is H or CN;

where

R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are the same or different and each is: H; orC₁-C₁₀ alkyl or aralkyl which may optionally contain 1 to 4 oxygen,nitrogen or sulphur atoms in a straight or branched chain; or R^(c)where R^(c) is a group to modify solubility, or a group to modifyelectronic and/or spectral properties, or a functional linking group; or—R^(a)R^(c) wherein R^(a) and R^(c) are herein defined,

R¹⁰ and R¹¹; and/or R¹³ and R¹¹; and/or R¹² and R¹⁴ may be linked toform an extended ring system, carbocyclic or heterocyclic, aromatic oraliphatic which is unsubstituted or substituted as above,

Z is aryl which is unsubstituted or substituted by: a group to modifysolubility; or a group to modify electronic and/or spectral properties;or a functional group for linking to another component, provided that Zis not 2-carboxy-3,4,5,6-tetrachloro phenyl;

 provided that NR¹³R¹⁴ is different from NR¹R²;

where

A is N or CH

A¹, A²A³ and A⁴ are the same or different and each is: H; or C₁-C₁₀alkyl or aralkyl which may optionally contain 1 to 4 oxygen, nitrogen orsulphur atom in a straight or branched chain; or R^(c) where R^(c) is agroup to modify solubility, or a group to modify electronic and/orspectral properties, or a functional linking group; or —R^(a)R^(c) whereR^(a) and R^(c) are herein defined;

where

n is 0, 1 or 2, B¹ is H, C₁-C₁₀ alkyl, aryl or aralkyl eitherunsubstituted or substituted by a group to modify solubility or a groupto modify electronic and/or spectral properties, or a functional linkinggroup, Cl, Br or F,

B² and B³ are the same or different and are selected from C₁-C₁₀ alkyl,aryl or aralkyl either unsubstituted or substituted by a group to modifysolubility or a group to modify electronic and/or spectral properties,or a functional linking group,

B¹ and B² and/or B¹ and B³ and/or B² and/or B³ may be linked to form anextended ring system, carbocyclic or heterocyclic, aromatic or aliphaticwhich is unsubstituted or substituted by a group to modify solubility,or a group to modify electronic and/or spectral properties, or afunctional linking group;

where

D, D¹, D² and D³ are the same or different and each is: H; or C₁-C₁₀alkyl or aralkyl which may optionally contain 1 to 4 oxygen, nitrogen orsulphur atom in a straight or branched chain; or R^(c) where R^(c) is agroup to modify solubility, or a group to modify electronic and/orspectral properties, or a functional linking group; or —R^(a)R^(c) whereR^(a) and R^(c) are herein defined;

where

E¹ is H, C₁-C₁₀ alkyl, aryl or aralkyl either unsubstituted orsubstituted by a group to modify solubility or a group to modifyelectronic and/or spectral properties, or a functional linking group,Cl, Br or F,

E² and E³ are the same or different and are selected from C₁-C₁₀ alkyl,aryl or aralkyl either unsubstituted or substituted by a group to modifysolubility or a group to modify electronic and/or spectral properties,or a functional linking group,

E¹ and E² and/or E¹ and E³ and/or E² and/or E³ may be linked to form anextended ring system, carbocyclic or heterocyclic, aromatic or aliphaticwhich is unsubstituted or substituted by a group to modify solubility,or a group to modify electronic and/or spectral properties, or afunctional linking group;

where

R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³ and R²⁴ are the same ordifferent and each is: H; or C₁-C₁₀ alkyl or aralkyl which mayoptionally contain 1 to 4 oxygen, nitrogen or sulphur atoms in astraight or branched chain; or R^(c) where R^(c) is a group to modifysolubility, or a group to modify electronic and/or spectral properties,or a functional linking group; or —R^(a)R^(c) wherein R^(a) and R^(c)are herein defined,

R¹⁶ and R¹⁷; and/or R²¹ and R²²; and/or R¹⁵ and R¹⁸ and/or R¹⁶ and R¹⁹;and/or R²⁰ and R²³; and/or R²¹ and R²⁴ may be linked to form an extendedring system, carbocyclic or heterocyclic, aromatic or aliphatic which isunsubstituted or substituted as above.

By virtue of the reactive group R¹, the dyes of the present inventionmay be combined with target materials to form conjugates. Suitabletarget materials may include antibodies, antigens, proteins,carbohydrates, polysaccharides, lipids, nucleotides, nucleic acids,polymer particles or surfaces and glass beads and surfaces. Thus, forexample, the carboxyl group of R¹ may be converted to anN-hydroxysuccinimidyl ester for linking with an amino group of apolypeptide. These methods and the products resulting from them, eitherdye-labelled biomolecules or dyes immobilised on particulate or massivesurfaces, are envisaged as further aspects of the invention.

The main uses for the dyes of this invention are expected to be forlabelling biologically active molecules such as nucleotides, oligo- andpoly-nucleotides, nucleic acids and nucleic acid analogues (e.g. PNA),amino acids, peptides, proteins, antibodies, haptens, etc. for use inbiological detection systems. Alternatively, the dyes may be conjugatedto species which can direct the path of the dye within or aid entry toor exit from cells (live or dead); such as for example long alkylresidues to allow permeation of lipophilic membranes, or intercalatingspecies to localise a dye in a nucleus or other cellular enclavecontaining double-stranded DNA or in vitro applications involvingdouble-stranded nucleic acids. Dyes may also be immobilised onsubstrates such as particles or beads. The dyes may be involved in aresonance energy transfer system of the kind described in EPA 747 700.

The actual properties of the dyes used as detection labels vary. Theobserved property may simply be colour (based on light absorbance),luminescence or fluorescence (based on light emission) or otherproperties such as fluorescence lifetimes or degree of polarisation ofemitted light. The dyes may also be used outside biological detectionsystems. For example, a dye may be immobilised on a probe for use in achemical detection system e.g. a colour change, pH probe, or a chemicaldetector based on solvatochromism, electrochromism, thermochromism orphotochromism. Reactive dyes may also be useful in printing e.g. inorder to obtain improved substantivity effects.

There follow a number of example applications, which indicate a generalchemical strategy by which intermediate compounds of formula (I) areused to make various families of dyes. The lettering system used isconsistent within any particular application, but may not be consistentwith that used in the introduction above and the claims below.

Application 1: Green-fluorescent Coumarin Dyes

If X is simply —CN or an aryl substituent then the dyes are colourlesswith a blue fluorescence emission. To obtain yellow dyes with a greenfluorescence the moiety X is a benzimidazole, benzoxazole orbenzthiazole ring system, with or without additional substituents (e.g.halogen, sulphonic acid).

Application 2: Yellow and Orange Fluorescent Coumarin Dyes

Application 3: Red-fluorescent Benzopyrano-benzimidazo-pyridines

Substituents R in the benzimidazole ring may be H or any commonsubstituent, but most usefully could be —SO₃H to enhance watersolubility or a grouping capable of forming a linkage, e.g. —CO₂H, togive a bis-functional dye.

Application 4: Red-fluorescent Rhodamine Dyes

As shown above, such dyes may be symmetrical or unsymmetrical. There isthe problem of the additional carboxylic acid group in the meso phenylring, which should preferentially be esterified. Alternatively,sulphorhodamines or non-meso-carboxylated analogues could be considered:

Application 5: Near-infrared Absorptive Croconium Dyes

Use of this type of intermediate (i.e. with a meta-hydroxy group) isessential for the success of the reaction with croconic acid. If thedyes are fluorescent, then the emission will probably lie beyond therange of most fluorimeters, probably near 900 nm.

Application 6: pH Sensitive Photochromic Dyes

Application 7: pH Sensitive Green-black Fluorans

The open chain form of the dye is not fluorescent and is usually verydark in colour due to its multiple absorption band chromophore.

Application 8: Styryl Hemi-cyanines

Application 9: Leuco Dyes for Use as Enzyme Substrates

These are not dyes, as such, but a reduced, or leuco, form of thosealready listed which for example, may be chemically or enzymaticallyoxidised back to parent dye molecule. The dyes may be derivatisedslightly to increase their stability.

Application 10: Oxindigo Derivatives

These dyes can have large Stoke's shift (50-100 nm) emitting in the red,λ_(max)(Abs.) 510-530 nm, λ_(max) (Em.) 600-620 nm. They are alsoreported as being strongly fluorescent with high colour fastness.(Angew. Chem. Int. Ed. Engl. 1996, 35 (9) 1016), and therefore theability to sum multiple scans.

Application 11: Squarylium Dyes

Application 12: 5,10,15,20-Tetraarylporphyrin Dyes

with or without a central metal ion.

The following examples illustrate the invention. Examples 1 to 5, showsthe preparation of compounds according to formula (I). Examples 6 to 18show the use of intermediates of formula (I) in the preparation ofvarious dyes.

Examples of Intermediate (I) EXAMPLE 13-N-Ethyl-N-(2′methoxycarbonylethyl)-aminophenol (Ia) R¹=—CH₂CH₂CO₂Me,R²=Et, R³=R⁴=R⁵=R⁶=H

3-Ethylaminophenol (5 g, 36 mmol), methyl acrylate (10 g, 120 mmol) andacetic acid (10 ml) were heated at reflux for 5 hours. The excess methylacrylatelacetic acid was then removed under reduced pressure at atemperature no higher than 70° C. The residual oil was extracted intoethyl acetate (50 ml), washed with saturated sodium bicarbonate solution(2×50 ml), water (100 ml), dried over MgSO₄, filtered and evaporated todryness in vacuo. The product (Ia) was isolated as a brown oil (7.2 g,90%).

¹H NMR (300 MHz, CDCl₃) 1.20 (t, 3H, J=6.9 Hz), 2.63 (t, 2H, J=7.1 Hz),3.33 (q, 2H, J=6.9 Hz), 3.60 (t, 2H, J=7.5 Hz), 3.70 (s, 3H), 6.15 (d,1H, J=5.4 Hz), 6.18 (s, 1H), 6.27 (d, 1H, J=5.4 Hz), 7.04 (t, 1H, J=6.9Hz).

EXAMPLE 2 3-N-(2′Methoxvcarbonylethyl)-N-ethylamino)-2-methylphenol (Ib)R¹=—CH₂CH₂CO₂Me, R²=H, R³=Me, R⁴=R⁵=R⁶=H

3-Amino-2-methylphenol (1 g, 8.1 mmol), methyl acrylate (0.73 ml, 8.1mmol) and acetic acid (2 ml) were heated together at reflux for 5 hrsand then cooled to room temperature. The excess acetic acid was removedunder reduced pressure and the residue purified by column chromatography(silica, 1:3 ethyl acetate: 40-60 petroleum ether). The product (Ib) wasobtained as an off white solid (0.81 g, 48%).

¹H NMR (300 MHz, CDCl₃) 1.98 (s, 3H,), 2.66 (t, 2H, J=6.3 Hz), 3.49 (t,2H, J=6.3 Hz), 3.71 (s, 3H), 6.25 (dd, 2H, J=7.8 Hz), 6.97 (t, 1H, J=8.1Hz).

EXAMPLE 3Tetra-n-butylammonium-6-hydroxy-2-(N-2′methoxycarbonylethyl-benzenesulphonate(Ic)

2-Amino-6-hydroxybenzenesulfonic acid (236 mg, 1.25 mmol) andtetra-n-butylammonium hydroxide (1M solution in methanol, 1.25 ml, is1.25 mmol) were stirred together at room temperature for 1 hr. Themethanol was removed under reduced pressure to leave a yellow oil whichwas washed with ether (3×5 ml). To the yellow oil was added methylacrylate (0.5 ml, 5.5 mmol) and acetic acid (1 ml) and the mixtureheated at reflux for 48 hrs. Excess methyl acrylatelacetic acid wasremoved under reduced pressure and the residue purified by columnchromatography (silica: 20% methanol in ethyl acetate). The product (Ic)was obtained as an off white solid (50 mg, 7.3%), partially in the formof the tetrabutylammonium salt.

¹H NMR (300 MHz, d₆DMSO) 3.23 (t, 2H, J=5.7 Hz), 3.53 (t, 2H, J=5.7 Hz),3.60 (s, 3H), 5.87 (s, 1H), 6.01 (d, 1H, J=6.3 Hz), 7.10 (d, 1H, J=8.4Hz).

EXAMPLE 4 3-Acetoxy-N-ethyl-N-(2′methoxycarbonylethyl)-aniline (Id)R¹=—CH₂CH₂CO₂Me, R²=Et, R³=R⁴=R⁵=R⁶=H, OCOCH₃

3-N-Ethyl-N-(2′methoxycarbonylethyl)-aminophenol (Ia) (7.2 g, 30 mmol)and acetic anhydride (10 ml) were heated at 100° C. for 4 hours. Oncooling the reaction mixture was poured onto ice and the pH adjusted to4-5 with KOH solution (10%). The product was extracted intodichloromethane, dried over MgSO₄, filtered and evaporated to dryness invacuo. The product (Id) was isolated as a brown oil (7.4 g, 93%).

¹H NMR (300 MHz, CDCl₃). 1.14 (t, 3H, J=13.3 Hz), 2.28 (s, 3H), 2.60 (t,2H, J=7.4 Hz), 3.36 (q, 2H, J=7.2 Hz), 3.63 (t, 2H, J=7.4 Hz), 3.71 (s,3H), 6.34 (s, 1H), 6.39 (d, 1H, J=7.4 Hz), 6.52 (d, 1H, J=8.4 Hz), 7.20(t, 1H, J=8.3 Hz).

EXAMPLE 52-Acetoxy-4-(N-ethyl-N-(2′methoxycarbonylethyl)-aminobenzaldehyde (Ie)R¹=—CH₂CH₂CO₂Me, R²=Et, R³=R⁴=R⁵=H, R⁶=CHO, OCOCH₃

3-Acetoxy-N-ethyl-N-(2′methoxycarbonylethyl)-aniline (Id) (6.5 g, 24mmol) was dissolved in dimethylformamide (10 ml, 0.13 mol) and cooled tobelow 5° C. Phosphorus oxychloride (3 ml, 33 mmol) was added dropwisemaintaining the temperature below 10° C. The reaction mixture was heatedto 90° C. for 1.5 hours and then cooled to room temperature andcarefully poured onto ice (400 g). The pH was adjusted to 5 with theaddition of saturated sodium acetate solution and stirred for 3 hours.The product was extracted into dichloromethane (2×150 ml), washed withwater (2×150 ml), dried over MgSO₄, filtered and evaporated to drynessin vacuo. The product was purified by column chromatography (silica;ethyl acetate) and isolated as a pale yellow oil (4.1 g, 58%).

¹H NMR (300 MHz, CDCl₃) 1.18 (t, 3H, J=8.6 Hz), 2.38 (s, 3H), 2.60 (t,2H, J=7.1 Hz), 3.45 (q, 2H, J=7.2 Hz), 3.69 (t, 2H, J=7.2 Hz), 3.76 (s,3H), 6.56 (s, 1H), 6.59 (d, 1H, J=9 Hz), 7.69 (d, 1H, J=9 Hz), 9.77 (s,1H).

Examples of Green Fluorescent Coumarin Dyes (II)—Application 1 EXAMPLE 6

Benzimidazol-2′-yl-7-Methyl-N(2″methoxycarbonylethyl)amino)iminocoumarin (IIa)

2-Acetoxy-4-(N-ethyl-N-(2′methoxycarbonylethyl)-aminobenzaldehyde (Ie)(2.18 g (60% purity), 4.5 mmol), 2-cyanomethylbenzimidazole (0.72 g, 4.5mmol), piperidine (0.45 ml) and dimethylformamide (5 ml) were stirredtogether at room temperature for 4 hours. Methanol (15 ml) was added andthe product filtered off, washed with methanol and dried. The productwas isolated as a yellow solid (1.2 g, 68%).

λ_(max) (DMF) 422 nm.

Fluorescence (DMF) λ_(ex) 422 nm; λ_(em) 490 nm; Stokes Shift 68 nm.

¹H NMR (300 mHz, d₆DMSO), 1.10 (t, 3H, J=7.1 Hz), 2.62 (t, 2H, J=6.9Hz), 3.42 (q, 2H, J=6.6 Hz), 3.61 (s, 3H), 3.66 (t, 2H, J=6.6 Hz), 6.43(s, 1H), 6.62 (d, 1H, J=8.7 Hz), 7.17 (m, 2H), 7.45 (d, 1H, J=8.7 Hz),7.62 (broad s, 2H), 8.36 (broad s, 1H).

Benzimidazol-2′-yl-7-(N-carboxyethyl-N-ethylamino) imino coumarin (IIb)

Ethanolic potassium hydroxide (2 ml (5%)) was added to a solution of thecoumarin (IIa) (0.20 g, 5.13 mmol) in ethanol (10 ml). The reactionmixture was heated at reflux for 10 mins at which point a furtheraliquot of 5% ethanolic KOH (4 ml) was added and the mixture was heatedat reflux for a further 15 mins. On cooling to room temperture the pHwas adjusted to pH 5 with the addition of 10N hydrochloric acid. Theethanol was removed under reduced pressure and the residue dissolved into dichloromethane (20 ml). The product was extracted into 1M NaOHsolution (50 ml) and then reprecipitated by adding 10N hydrochloricacid, the solid was filtered off, washed with water and dried. Theproduct (IIb) was obtained as a bright yellow solid (90 mg, 47%).

λ_(max) (DMF) 425 nm.

Fluorescence (DMF) λ_(ex) 425 nm; λ_(em) 490 nm; Stokes Shift 65 nm.FAB-MS m/z=found 399 (MNa⁺), 377 (MH⁺) [theoretical (C₂₁H₂₀N₄O₃) 376].

¹H NMR (300 MHz. d₆DMSO), 1.13 (t, 3H, J=6.6 Hz,), 2.57 (t, 2H), 3.53(q, 2H, J=7.5 Hz), 3.69 (t, 2H, J=7.5 Hz), 6.70 (s, 1H), 6.84 (d, 1H,J=11.0 Hz), 7.16 (m, 1H), 7.24 (m, 1H), 7.61 (m, 1H), 7.64 (m, 1H) 7.72(d, 1H, J=9.2 Hz), 8.93 (s, 1H).

Benzimidazol-2′-yl-7-(N-carboxyethyl-N-ethylamino) coumarin (IIc)

Imino coumarin (IIa) (0.5 g, 1.28 mmol), water (45 ml) and conc. HCl (5ml) were heated at 100° C. for 2 hours, over which time the dyedissolved and reprecipitated. The product was filtered off, washed withmethanol and dried. The product was isolated as a bright yellow solid(0.3 g, 63%).

λ_(max) (DMF) 438 nm.

Fluorescence (DMF) λ_(ex) 438 nm; λ_(em) 495 nm; Stokes Shift 57 nm.

¹H NMR (300 MHz, d₆DMSO), 1.10 (t, 3H, J=6.9 Hz), 2.57 (t, 2H, J=7.1Hz), 3.52 (q, 2H, J=7.2 Hz), 3.68 (t, 2H, J=7.2 Hz), 6.70 (s, 1H), 6.84(d, 1H, J=11.4 Hz), 7.16 (m, 2H), 7.60 (m, 2H), 7.73 (d, 1H, J=9.3 Hz),8.96 (s, 1H).

3-(5′,6′-disulphobenzimidazol-2′-yl-7-(N-carboxyethyl-N-ethylamino)coumarin (IId)

To coumarin (IIc) (90 mg, 0.24 mmol) was added chlorosulphonic acid (0.5ml). The reaction mixture was stirred at 40° C. for 5 hrs, cooled, andthen carefully quenched over ice. The orange precipitate was filteredoff, washed with water, ethyl acetate and dried. The dye was purified byHPLC (RP C₁₈; 80:20 water:ethanol). The product (IId) was obtained as aorange solid (5 mg, 3.6%).

μ_(max) (1:1 MeOH:H₂O) 370 nm.

Fluorescence; none detected.

¹H NMR (300 MHz, d₆DMSO) 1.21 (t, 3H), 3.4 (broad t, 2H), 3.61 (q, 2H,),4.04 (t, 2H), 6.91 (d, 1H), 7.59 (d, 1H), 7.70 (s, 2H), 7.98 (s, 1H),8.92 (s, 1H).

It was noted that if coumarin dye (IId) was treated with methanolcontaining a catalytic quantity of sulphuric acid the non-fluorescentdye (IId) was converted to the highly fluorescent dye (IIe);

λ_(max) (1:1 MeOH:H₂O) 424 nm.

Fluorescence (1:1 MeOH:H₂O) λ_(ex) 406 nm; λ_(em) 480 nm;

Stokes Shift 74 nm.

EXAMPLE 7

3-(6′chlorobenzimidazol-2′-yl-7(N-ethyl-N-(2″methoxycarbonylethyl)amino) imino coumarin—(IIf)

2-Acetoxy-4-(N-ethyl-N-(2′methoxycarbonylethyl)-amino)-benzaldehyde (Ie)(1 g, 3.4 mmol), 2-cyanomethyl-6-chloro-benzoxazole (0.66 g, 3.4 mmol),piperidine (0.6 ml) and dimethylformamide (2.5 ml) were stirred togetherat room temperature for 5 hours. The crude dye was precipitated from thereaction mixture by adding ether and then filtered off, washed withether and dried. The dye was purified by column chromatography (silica;1:4 methanol:ethyl acetate). The product (IIf) was obtained as anyellow/brown solid (0.5 g, 36%).

λ_(max) (MeOH) 454 nm.

Fluorescence (MeOH) λ_(ex) 453 nm; λ_(em) 494 nm; Stokes Shift 41 nm.

¹H NMR (300 MHz, d₆DMSO), 1.10 (t, 3H, J=7.1 Hz), 2.64 (t, 2H, J=7.1Hz), 3.47 (q, 2H, J=6.9 Hz), 3.61 (s, 3H), 3.68 (t, 2H, J=7.1 Hz), 6.50(s, 1H), 6.65 (d, 1H, J=6.6 Hz), 7.30 (d, 1H, J=9.0 Hz), 7.53 (d, 1H,J=9.0 Hz), 7.76 (d, 1H, J=9 Hz), 8.35 (s, 1H).

Examples of Yellow and Orange Fluorescent Coumarins Dyes (II) andIntermediates Used in Their Preparation—Application 2 EXAMPLE 8

7(N-Ethyl-N-(2′methoxycarbonylethyl)-amino)-4-hydroxy coumarin (IIh)

3-N-Ethyl-N-(2′methoxycarbonylethyl)-aminophenol (Ia), malonic acidbis-2,4,6-trichlorophenol ester and toluene (40 ml) were heated atreflux for 4 hours. On cooling the product was filtered off, washed withcold acetone and dried. The product (IIh) was obtained as a pale yellowsolid (4.1 g, 63%).

¹H NMR (300 MHz, d₆DMSO) 1.07 (t, 3H, J=7.0 Hz), 2.60 (t, 2H, J=7.1 Hz),3.43 (q, 2H, J=7.0 Hz), 3.60 (s, 3H), 3.62 (t, 2H, J=7.0 Hz), 5.26 (s,1H), 6.49 (s, 1H), 6.67 (d, 1H, J=8.7 Hz), 7.54 (d, 1H, J=8.0 Hz).

4-Chloro-7-(N-ethyl-N-(2′methoxycarbonylethyl)-aminocoumarin-3-carboxaldehyde (IIi)

7-(N-Ethyl-N-(2′methoxycarbonylethyl)-amino)-4-hydroxy coumarin (IIh)(2.5 g, 8.6 mmol) and dimethylformamide (15 ml) were cooled to below 5°C. Phosphorus oxychloride (1 ml) was added dropwise mantaining thetemperature below 10° C. The reaction mixture was gently warmed to 35°C. for 3 hours. On cooling the reaction mixture was poured onto ice andstirred for one hour. The product was extracted into ethyl acetate (50ml), washed with water (100 ml), dried over MgSO₄, filtered andevaporated to dryness in vacuo. The product was purified by columnchromatography. (silica; ethyl acetate). The product (IIi) was isolatedas an orange solid (2.18 g, 75%).

¹H NMR (300 MHz, d₆DMSO), 1.06 (t, 3H, J=7.0 Hz), 2.66 (t, 2H, J=7.1Hz), 3.53 (q, 2H, J=7.0 Hz), 3.61 (s, 1H), 3.73 (t, 2H, J=7.0 Hz), 6.67(s, 1H), 6.93 (d, 1H), 7.82 (d, 1H), 10.07 (s, 1H).

EXAMPLE 9

3-(N-ethyl-N-(2′methoxycarbonylethyl)-amino)-6oxo-6H-[1]Benzopyrano[3′,4′:5,6]pyrido[1,2a]benzimidazole-8-carbonitrile,(IIi)

Coumarin (IIi) (0.5 g, 4.8 mmol), 2-cyanomethylbenzimidazole (0.23 g,4.8 mmol) and acetic anhydride (5 ml) were heated together at 100° C.for 4 hours. On cooling the product was precipitated out of the reactionmixture with ether. The product was filtered off, washed with ether anddried. The product (IIj) was obtained as an orange solid (0.5 g, 74%).

λ_(max) (MeOH) 490 nm.

Fluorescence (MeOH) λ_(ex) 490 nm; λ_(em) 540 nm; Stokes Shift 50 nm.

¹H NMR (300 MHz, d₆DMSO), 1.16 (t, 3H, J=6.8 Hz), 2.70 (t, 2H, J=6.9Hz,), 3.56 (q, 2H, J=6.9 Hz), 3.63 (s, 3H), 3.76 (t, 2H, J=7.4 Hz), 6.81(s, 1H), 6.88 (d, 1H, J=9 Hz), 7.41 (t, 1H, J=7.8 Hz), 7.63 (t, 1H,J=7.5 Hz), 7.98 (d, 1H, J=8.1 Hz), 8.13 (d, 1H, J=8.4 Hz), 8.15 (d, 1H,J=9.6 Hz), 8.51 (s, 1H).

3-(2-carboxyethyl-N-ethylamino)-6-oxo-6H-[1]Benzopyrano[3′,4′:5,6]pyrido[1,2a]benzimidazole-8-carbonitrile,(IIk)

Coumarin (IIj) (50 mg, 0.11 mmol) and ethanolic KOH (0.5 ml (5%)) wereheated at reflux for 10 mins. On cooling the pH was adjusted to 7 withhydrochloric acid. The product was filtered off, washed with water anddried. The product was obtained as an orange solid (50 mg, 106% —stillcontaminated with inorganic salts).

λ_(max) (MeOH) 456 nm.

Fluorescence (MeOH) λ_(ex) 487 nm; λ_(em) 540 nm; Stokes Shift 50 nm.

¹H NMR (300 MHz, d₆DMSO), 1.16 (t, 3H, J=6.8 Hz), 2.58 (t, 2H, J=7.4Hz), 3.43 (q, 2H, J=6.9 Hz), 3.60 (t, 2H), 6.33 (s, 1H), 6.39 (d, 1H,J=8.1 Hz), 6.63 (d, 1H, J=8.4 Hz), 7.07 (d, 1H, J=8.7 Hz), 7.16 (t, 1H,J=8.0 Hz), 7.51 (t, 1H, J=7.8 Hz), 7.90 (d, 1H, J=7.8 Hz), 8.6 (s, 1H).

3-(N-ethyl-N-(2′methoxycarbonylethyl)-amino)-6-oxo-6H-[1]Benzopyrano[3′,4′:5,6]pyrido-3-sulphobutyl-[1,2a]benzimidazolium-8-carbonitrile(IIl)

Coumarin (IIj) (50 mg, 0.11 mmol); 1,4-butanesultone (17.5 mg, 0.13mmol) and butyronitrile (0.5 ml) were heated together at reflux for 5hours. On cooling the dye was precipitated out of the reaction mixturewith ethyl acetate and filtered off. The dye was purified by preparativeTLC (RP C₁₈; 3:2 methanol:water). The product was obtained as an orangesolid (16 mg, 25%).

MALDI-TOF-MS m/z=; found 576 (M); [theoretical (C₂₉H₂₈N₄SO₇) 576]

EXAMPLE 10

3-(benzimidazol-2′-yl)-4-cyano-7-(N-ethyl-N-(2″methoxycarbonylethyl)amino) coumarin—(IIm) and3-(benzimidazol-2′-yl)-4-cyano-7-(N-carboxyethyl-N-ethylamino) coumarin(IIn)

Imino coumarin (IIa) (1 g, 2.56 mmol) was dissolved in dimethylformamide(40 ml) and warmed to 40° C. Sodium cyanide (0.25 g, 5.1 mmol) in water(10 ml) was added and the solution stirred at 40° C. for 2 hours. Thesolution was then cooled to 10° C. and bromine (0.82 g, 5.1 mmol) added.Stirring was continued at this temperature for a further 2 hours. Thesolution was poured onto water (200 ml) and the precipitated productfiltered off, washed with water and dried in a vacuum desiccator. Theproduct was obtained as a red powder (0.67 g, 63%). The dye (50 mg) waspurified by column chromatography (silica, 1:1 ethylacetate:40-60petroleum ether) to give the methyl ester containing dye (IIm) (6 mg(12%)) and the free carboxylic acid containing dye (IIn) (3 mg (6%)).

Dye (IIm)

λ_(max) (MeOH) 490 nm.

Fluorescence (MeOH) λ_(ex) 490 nm; λ_(em) 597 nm; Stokes Shift 107 nm.

¹H NMR (300 MHz, d6 DMSO) 1.1 (t, 3H), 2.67 (t, 2H), 3.57 (q, 2H), 3.62(s, 3H), 3.73 (t, 2H), 6.82 (s, 1H), 7.01 (d, 1H), 7.21 (m, 2H), 7.26(t, 2H), 7.70 (m, 3H).

Dye (IIn)

λ_(max) (MeOH) 486 nm.

Fluorescence (MeOH) λ_(ex) 492 nm; λ_(em) 597 nm; Stokes Shift 105 nm.

¹H NMR (300 MHz, d₆ DMSO) 1.09 (t, 3H), 2.67 (t, 2H, J=6.9 Hz), 3.57 (q,2H), 3.73 (t, 2H, J=6.9 Hz), 6.82 (s, 1H), 7.01 (d, 1H, J=9.3 Hz), 7.21(m, 2H), 7.26 (t, 2H), 7.70 (m, 3H).

Examples of Red Fluorescent Benzopyranobenzimidazopyridine Dyes(III)—Application 3 EXAMPLE 11

Benzopyranobenzimidazopyridine Dye (IIIa)

A mixture of coumarin (IIa) (2.0 g, 5.13 mmol) and malononitrile (0.35g, 5.30 mmol) in 1-butanol was heated at reflux for 4 hours. The mixturewas concentrated to ca. 10 ml and left to stand overnight during whichtime the product crystallised out as a dark red solid. The product wasfiltered off, washed with butanol and dried (1.97 g, 87%). The dye(IIIa) was purified by recrystallisation from 1-butanol.

λ_(max) (DMF) 541 nm.

Fluorescence (DMF) λ_(ex) 541 nm; λ_(em) 574 nm; Stokes Shift 33 nm.

FAB-MS m/z; found 440 (MH⁺); [theoretical (C₂₅H₂₁N₅O₃) 439].

¹H NMR (300 MHz, d₆DMSO) 1.16 (t, 3H, J=6.6 Hz), 2.68 (t, 2H), 3.56 (q,2H), 3.61 (s, 3H), 3.74 (t, 2H), 6.78 (s, 1H), 6.92 (d, 1H, J=6.9 Hz),7.37 (m, 2H, J=7.4 Hz), 7.72 (d, 2H, J=8.1 Hz), 8.64 (d, 1H, J=7.2 Hz),8.68 (s, 1H).

Functionalised Benzopyranobenzimidazopyridine Dye (IIIb)

Imino benzopyranobenzimidazopyridine (IIIa) (200 mg, 0.45 mmol) washeated in a mixture of water (20 ml) and 10N hydrochloric acid (2 ml) at100° C. for 2 hours. The solution was then cooled and the pH adjusted to5 with the addition of potassium hydroxide solution. The water wasremoved under reduced pressure and the product washed with methanol,water and then dried. The product (IIIb) was obtained as a purple solid(67 mg, 35%).

λ_(max) (DMF) 545 nm.

Fluorescence (DMF) λ_(ex) 545 nm; λ_(em) 578 nm; Stokes Shift 33 nm.

¹H NMR (300 MHz, d₆DMSO) 1.16 (t, 3H), 2.63 (t, 2H), 3.56 (q, 2H), 3.74(t, 2H), 6.97 (s, 1H), 7.14 (d, 1H), 7.41 (m, 2H), 7.74 (d, 2H), 8.66(d, 1H), 8.71 (s, 1H).

Sulphonated, Functionalised Benzopyranobenzimidazopyridine Dye (IIIc)

Benzopyranobenzimidazopyridine dye (IIIb) (1 g) was stirred in 25% oleumat room temperature for 3 hours, and the solution was poured onto iceand neutralised with NaOH. The solution was evaporated to dryness and aportion of the sodium sulphate/dye mixture (6.5 g) was taken and stirredin water (50 ml) at room temperature for 30 minutes and thencentrifuged. The supernatant was discarded and the washing processrepeated with a small amount of water. The resultant solid (60 mg) wasdried in a vacuum desiccator. This showed a single red fluorescent spoton t.l.c.

λ_(max) (H₂O) 536 nm.

Fluorescence (H₂O) λ_(ex) 536 nm; λ_(em) 564 nm; Stokes Shift 28 nm.Electrospray (−ve ion)-MS m/z; found 584; [theoretical (C₂₄H₁₆N₄S₂O₁₀)584].

Using milder sulphonation conditions and purification of the product byHPLC (RP C₁₈, 33% methanol in water), both the methyl ester (IIId) andthe free acid (IIIe) forms of the monosulphonatedbenzopyranobenzimidazopyridine dye were also isolated.

(IIId)—Methyl ester

MALDI-TOF-MS m/z; found 519 (M⁺); [theoretical (C₂₅H₂₀N₄SO₇) 520].

¹H NMR (300 MHz, d₆DMSO), 1.17 (t, 3H, J=6.9 Hz), 2.71 (t, 2H), 3.37 (q,2H), 3.63 (s, 3H), 3.80 (t, 2H), 6.78 (s, 1H), 7.03 (d, 1H, J=7.2 Hz),7.63 (d, 1H, J=8.4 Hz), 7.71 (d,1H, J=8.4 Hz), 7.84 (d, 1H), 8.85 (s,1H), 8.97 (s, 1H).

(IIIe)—Free acid

MALDI-TOF-MS m/z; found 504 (M⁺); [theoretical(C₂₄H₁₇N₄SO₇) 505].

¹H NMR (300 MHz, d₆DMSO), 1.17 (t, 3H), 2.27 (t, 2H), 3.63 (m, 4H), 6.67(s, 1H), 7.02 (d,1H), 7.62 (d,1H, J=8.4 Hz), 7.70 (d, 1H, J=8.4 Hz),7.80 (d, 1H, J=9.6 Hz), 8.81 (s, 1H), 8.96 (s, 1H).

EXAMPLE 12

N-Benzoylimino Benzopyranobenzimidazopyridine (IIIf)

Imino benzopyranobenzimidazopyridine dye (IIIa) (200 mg, 0.46 mmol) andbenzoyl chloride (1 g, 7.12 mmol) in pyridine (10 ml) were heated at 80°C. overnight. The resultant mixture was poured into water (200 ml) andstirred at room temperature for 1 hr and then extracted withdichloromethane. The organic phase was washed with 10% sodiumbicarbonate and concentrated in vacuo. Purification of the residue byflash column chromatography (Silica: dichloromethane to 20%acetone/dichloromethane) afforded the desired product. The product(IIIf) was obtained as a dark red solid (180 mg, 73%).

λ_(max) (DMF) 559nm.

Fluorescence (DMF) λ_(ex) 559nm; λ_(em) 596 nm; Stokes Shift 37 nm.

FAB-MS m/z; found 566 (MNa+), 543 (M⁺); [theoretical (C₃₂H₂₅N₄O₄) 543).

N-Benzoylimino Benzopyranobenzimidazopyridine (IIIg)

Hydrolysis of dye (IIIf) under acidic conditions (dilute hydrochloricacid, 90° C., 2 h) afforded the free carboxylic acid containingN-benzoylimino-benzopyranobenzimidazopyridine dye (IIIg).

λ_(max) (DMF) 552 nm.

Fluorescence (DMF) λ_(ex) 552 nm; λ_(em) 580 nm; Stokes Shift 28 nm.

FAB-MS m/z; found 530 (MH⁺); [theoretical (C₃₁H₂₃N₅O₄) 529].

Example of Red-fluorescent Rhodamine Dyes (IV)—Application 4 EXAMPLE 13.

Monofunctionalised Rhodamine Dye (IVa)

3-N-Ethyl-N-(2′methoxycarbonylethyl)-aminophenol (Ia) (0.88 g, 4 mmol),2-(4′-N,N-diethylamino-2′-hydroxybenzoyl)benzoic acid (1.13 g, 4 mmol)and concentrated sulphuric acid (15 ml) were stirred together at roomtemperature for 24 hrs. The reaction mixture was then slowly neutralisedwith sodium hydroxide solution (20%) until the rhodamine dye started tocrystallise out of solution. The dye was filtered off and dried. The dyewas purified by column chromatography (silica; 1:1 chloroform/methanol,finally dissolving dye in chloroform and filtering to remove thesilica). The product (IVa) was obtained as a magenta powder (0.02 g,1%).

λ_(max) (MeOH) 544 nm.

Fluorescence (MeOH) λ_(ex) 547 nm; λ_(em) 570 nm; Stokes Shift 23 nm.

FAB-MS; found 487; [theoretical (C₂₉H₃₁N₂O₅) 487].

¹H NMR (300 MHz, CD₃OD) 1.28 (t, 9H, J=6.0 Hz), 2.53 (t, 1H, J=7.2 Hz),3.66 (m, 6H, J=7.5 Hz), 3.84 (t, 1H, J7.5 Hz), 7.00 (m, 4H), 7.26 (m,3H), 7.62 (m, 2H), 8.08 (d, 1H, J=4.5 Hz).

Examples of pH, Sensitive Photochromic Dyes (VI) and Basic Dye Forms(VII)—Application 6 EXAMPLE 14

4-(N-ethyl-N-(2′methoxycarbonylethyl)-amino)benzaldehyde (1 g, 0.40mmol), Fischer's base (0.6 g, 0.40 mmol), triethylamine (0.5 ml) andethanol (10 ml) were heated together at reflux for 4 hours. On coolingthe product crystallised out of solution and was filtered off, washedwith methanol and dried. The product was isolated in the colourlessspiro form (VIa) (0.7 g, 50%). It was noted that the non-fluorescentmerocyanine form (VIa open) could be switched to the fluorescent basicdye form (VIIa) by adding acid to a solution of dye in methanol.

Merocyanine Form (VIa)

λ_(max) (MeOH) 550 nm, E_(max) 37,677 l.mol⁻¹.cm⁻¹.

Fluorescence (MeOH) λ_(ex) 563 nm;λ_(em) 574 nm; Stokes Shift 11nm,—negligible fluorescence.

Basic Form (VIIa)

λ_(max) (MeOH) 544 nm, E_(max)71,968 l.mol⁻¹ .cm⁻¹.

Fluorescence (MeOH) λ_(ex) 569 nm; λ_(em) 581 nm; Stokes Shift 12 nm,

¹H NMR Basic dye (VIIa), (300 MHz, CD₃OD), 1.24 (t, 3H, J6.9 Hz), 1.76(s, 6H), 2.70 (t, 2H, J=7.2 Hz), 3.56 (q, 2H, J=6.9 Hz), 3.69 (s, 3H),3.78 (t, 2H, J=6.9 Hz), 3.83 (s, 3H), 6.18 (s, 1H), 6.52 (d, 1H, J=9.3Hz.), 7.17 (broad d) 7.41 (m, 4H), 7.81 (d, 1H, J=9.9 Hz), 8.57 (d, 1H,J=14.7 Hz).

Photochromic Dye (VIb)

2-Acetoxy-4-(N-ethyl-N-(2′methoxycarbonylethyl)-amino)-benzaldehyde (Ie)(0.5 g, 1.7 mmol), 1-ethyl-2,3,3-trimethyl-indolium-5-sulphonate (0.46g, 1.7 mmol), triethylamine (0.25 ml) and ethanol (10 ml) were heated atreflux for 5 hours. On cooling the ethanol was removed under reducedpressure to leave a magenta tar which was triturated with ether to givea sticky magenta solid. The dye was purified by column chromatography(silica; 30% methanol in ethyl acetate). The product (VIb) was obtainedas a purple solid (0.11 g, 11.4%). In order to convert dye (VIb) to thebasic dye form (VIIb) a small sample of the dye was dissolved inmethanol containing hydrochloric acid. The dye was then reprecipitatedby adding ether, filtered off and dried.

(VIIb) λ_(max) (MeOH) 554 nm.

Fluorescence (VIIb) (MeOH) λ_(ex) 544 nm; λ_(em) 577 nm;

Stokes Shift 33 nm.

¹H NMR (VIb) (300 MHz d₆DMSO), 1.14 (broad, 3H), 1.34 (broad, 3H), 1.69(s, 6H), 2.67 (broad, 2H), 3.48 (broad, 2H), 3.62 (s, 3H), 3.70 (broad,2H), 4.38 (broad), 6.29, (s, 1H), 6.53 (d, 1H,), 7.23 (d, 1H7.59 (d,1H), 7.71 (d, 1H), 7.90 (s, 1H,), 8.01 (d, 1H), 8.41 (d, 1H).

Example of pH Sensitive Green-Black Floran Dye (XIII)—Application 7EXAMPLE 15

Green-Black Floran Dye (VIIIb)

Phthalic anhydride (2.66 g, 0.018 mol) was added to a stirred solutionof 3-N-ethyl-N-(2′methoxycarbonylethyl)-aminophenol (Ia) (4 g, 0.018mol) in 5 ml chlorobenzene, the resulting solution was then heated at100° C. for 8 hours. The chlorobenzene solvent was removed on a rotaryevaporator yielding a red tar, which TLC showed to contain significantquantities of unreacted material (yield 6.57 g). This was purified bydissolving the solid in a dilute solution of sodium hydroxide, followedby extraction with dichloromethane. The product remained in the aqueousphase and was then extracted into dichloromethane after acidifiying thesolution. Evaporation of the solvent gave (VIIIa) (1.2 g; 18%).

Intermediate (VIIa) (0.3 g, 1.1 mmol) was dissolved in conc. H₂SO₄ (4ml) and 3-dimethylaminophenol (0.15 g, 1.1 mmol) was added in portionsover 15 minutes. The solution was then stirred at room temperature for65 hours. TLC analysis of the solution showed it to contain 3components.

The acidic solution (˜3 ml) was poured onto a 20 g water/ice mixture andslowly neutralised with concentrated sodium hydroxide, and the resultantfluoran lactone species was extracted at ˜pH 6 into 2×100 ml portions ofdichloromethane. The extracts were dried and evaporated under reducedpressure. This gave (VIIIb) in 1% yield. The visible absorption spectrumof the dye showed λ_(max)=530 nm under acid condition, the peakdisappearing (reversibly) under basic conditions due to lactoneformation.

The mass spectrum of the product was consistent with the assignedstructure (FAB: found m/Z=473, with weaker peak at m/Z=495; C₂₇H₂₄N₂O₆requires M+1=473, M+23=495).

Example of Styryl hemi-Cyanines (IX)—Application 8 EXAMPLE 16

3- Anilinoacrylaldehyde anil (0.25 g, 1.1 mmol) and acetic anhydride (1ml) were heated together at 40° C. for 30 mins.3-N-Ethyl-N-(2′methoxycarbonylethyl)-aminophenol (Ia) (0.5 g, 2.3 mmol)and acetic acid (5 ml) were then added and the mixture was warmed to 40°C. and stirred for 1 hr. On cooling ether (100 ml) was added and thesolvents were then decanted off to leave a cyan coloured tar.

λ_(max) (MeOH) 660 nm.

Electrospray-MS (+ve ion); found 483 (M⁺); [theoretical (C₂₇H₃₅N₂O₆)484].

Examples of Leuco Dyes for Use as Enzyme Substrates—Application 9EXAMPLE 17

Benzopyranobenzimidazopyridine Leuco Dye (a)

Sodium borohydride (excess) was added to a solution ofbenzopyranobenzimidazopyridine dye (IIIa) (0.5 g) indichloromethane/methanol (60/10 ml). After 10 mins, water (50 ml) and10N hydrochloric acid (1 drop) was added and the mixture was stirred fora further 10 mins in order to destroy the excess sodium borohydride. Thepale yellow solid (a) which formed was filtered off and dried overnightin vacuo.

Benzopyranobenzimidazopyridine Leuco Dye (b)

Sodium borohydride (at least 5 fold excess) was added to a solution ofthe benzopyranobenzimidazopyridine dye (IIIb) (1 g) in methanol (100ml). After the effervescence had ceased (ca. 10 min) the solution wasconcentrated in vacuo and then dried overnight in vacuo to afford thecrude leuco dye (b), 1.35 g.

In both of the above cases treatment of a solution of the leuco dye inDMF with a solution of chloranil in DMF regenerated the colour (andhence the absorption spectrum) of the parent dye.

Examples of squarylium (XI)—Application 11 EXAMPLE 18

Unsymmetrical squarylium dye (XI)

Dimethyl squarate (0.50 g) and Fischer's base (0.61) were dissolved inmethanol (15 ml) containing sodium methoxide (0.3 g) and the solutionstirred at 30° C. for 5 hours. To the solution was added n-butanol (40ml), toluene (20 ml), 3-N-ethyl-N-(2′methoxycarbonylethyl)-aminophenol(Ia) (0.79 g) and 5 drops of concentrated hydrochloric acid, and themixture heated under reflux for 4 hours. The solvent was then removedunder reduced pressure and the residue chromatographed over silica gelusing dichioromethane with increasing proportions of acetone as eluent.When the first blue band had been eluted the second blue band was elutedwith methanol, affording, after evaporation of the solvent, dye (XI)(0.45 g), which was characterised by mass spectrometry.

λ_(max) (MeOH) 628 nm.

Fluorescence (MeOH) λ_(ex) 628 nm; λ_(em) 648 nm; Stokes Shift 18 nm.

What is claimed is:
 1. A dye having the formula

wherein n represents 0, 1 or 2, B¹ represents H, or  represents C₁-C₁₀alkyl, aryl or aralkyl, each of which is either unsubstituted orsubstituted by a group to modify solubility, or a group to modifyelectronic and/or spectral properties, or a functional linking group, orCl, Br or F, B² and B³ independently represent C₁-C₁₀ alkyl, aryl oraralkyl, each of which is either unsubstituted or substituted by a groupto modify solubility, or a group to modify electronic and/or spectralproperties, or a functional linking group, B¹ and B² and/or B¹ and B³may be linked to form a one or two fused ring system, each ringcontaining 5 or 6 atoms, which is carbocyclic or heterocyclic, whereinsaid system can be either aromatic or aliphatic which is unsubstitutedor substituted by a group to modify solubility, or a group to modifyelectronic and/or spectral properties, or a functional linking group; R¹represents —R^(a)R^(b), where R^(a) is C₁-C₁₀ alkylene or aralkylenewhich may optionally contain 1 to 4 oxygen, nitrogen or sulphur atoms ina straight or branched chain, and R^(b) is carboxy, R², R³, R⁴ and R⁵independently represent H; or  C₁-C₁₀ alkyl or aralkyl which mayoptionally contain 1 to 4 oxygen, nitrogen or sulphur atoms in astraight or branched chain; or  R^(c) where R^(c) is a group to modifysolubility, or a group to modify electronic and/or spectral properties,or a functional linking group; or  —R^(a)R^(c) wherein R^(a) and R^(c)are herein defined as above, or R⁴—R⁵ or R²—R⁴ or R²-R³ are linked toform a single six membered ring system, which is carbocyclic orheterocyclic, wherein said system can be either aromatic or aliphaticwhich is unsubstituted or substituted by a group to modify solubility,or a group to modify electronic and/or spectral properties, or afunctional linking group.
 2. The dye of claim 1 which is in amerocyanine non-fluorescent form or in a basic fluorescent form.
 3. Aleuco-dye or reduced dye analogue of the dye as claimed in claim
 1. 4. Acomplex of a dye according to claim 1 with a biomolecule, wherein thecompound is covalently linked to the biomolecule through R¹.
 5. The dyeof claim 1 wherein said carboxy representing R^(b) is selected from thegroup consisting of acid, salt, ester, N-hydroxysuccinimide, activatedester and amide.